【Objective】To evaluate the clinical value of liquid chromatography with tandem mass spectrometry assay （LC- MS/MS） in determining hyperandrogenism and polycystic ovary syndrome （PCOS） by the examination of total testosterone（TT），in comparison of chemiluminesce immunoassay（CLIA）.【Methods】A total of 325 PCOS patients and 244 healthy controls women of reproductive age were included. The TT levels measured by LC-MS/MS and by CLIA were compared between patients with PCOS and controls.【Results】The TT levels measured by LC-MS/MS distinguished patients with PCOS from controls more notable than those by CLIA；as well as differentiated the population with hirsutism from non-hirsutism more dramatically than TT and corresponding FAI by CLIA. Furthermore，the TT level by LC-MS/MS was positively correlated with mFG scores，while there was no significant relation between TT by CLIA and mFG scores. Using TT by LC-MS/MS ≥ 1.85 nmol/L and TT by CLIA ≥ 2.39 nmol/L as the cut-off values for diagnosis of HA， respectively，there were more significant differences regarding the anthropological parameters and laboratory examination results between LC-MS/MS HA and CLIA HA group（P < 0.05）. ROC curves also showed that TT by LC-MS/MS had better diagnoses effect for PCOS than TT by CLIA.【Conclusions】The LC-MS/MS assay is more efficient and precise for detection of TT and diagnose of HA and PCOS than the CLIA assay. The LC-MS/MS assay has better clinical value for diagnosis of hyperandrogenemia and PCOS.